Isolation of Mitochondria from Animal Cells using the FOCUS™ Mitochondria Kit
OPTIONAL : Add appropriate protease inhibitor cocktail ( e . g . G - Biosciences’ Protease Arrest , Cat # 786 - 108 ) to SubCell Buffer - I just before use .
Use fresh cells only .
Pellet the harvested cells by centrifugation at ~ 800 x g for 1 minute .
Carefully remove and discard the supernatant .
Add 500µl of ice cold SubCell Buffer - I .
Gently vortex to suspend the cells and incubate on ice for 10 minutes .
Perform this lysis step on ice .
Using a narrow opening ( 20 gauge ) syringe needle , gently pull the suspension up and down 10 - 30 times .
( Alternatively , use Dounce homogenizer as described in the annotation below . )
Add 250µl 3X SubCell Buffer - II ( 350µl if Dounce homogenizer is used ) and mix by inverting .
This generates a 1X final concentration of SubCell Buffer - II .
Centrifuge the tube at 700x g for 10 minutes to pellet the nuclei .
Transfer the supernatant to a new tube .
Centrifuge supernatant at 12,000x g for 15 minutes .
The pellet contains mitochondria .
Transfer the supernatant ( cytosol fraction ) to a new tube .
Add 500µl 1X SubCell Buffer - II to the pellet , and centrifuge again at 12,000 x g for 5 minutes .
Discard the supernatant .
Suspend the mitochondrial pellet in 50 - 100µl .
Working Mitochondria Storage Buffer and keep the suspension on ice before downstream processing .
The suspension may be stored on ice for up to 48 hours .
